Structural basis for induced formation of the inflammatory mediator prostaglandin E2 Caroline Jegerscho¨ ld*†, Sven-Christian Pawelzik‡, Pasi Purhonen*, Priyaranjan Bhakat*§, Karina Roxana Gheorghe*, Nobuhiko Gyobu¶, Kaoru Mitsuokaʈ, Ralf Morgenstern**, Per-Johan Jakobsson†‡, and Hans Hebert*†

*Department of Biosciences and Nutrition, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, Novum, S-141 57 Huddinge, Sweden; ‡Department of Medicine, Rheumatology unit and Karolinska Biomic Center and **Institute of Environmental Medicine, Karolinska Insitutet, S-171 77 Stockholm, Sweden; and ¶Japan Biological Information Research Center, Japan Biological Informatics Consortium, and ʈBiological Information Research Center, National Institute of Advanced Industrial Science and Technology, Aomi 2-41-6, Koto-Ku, Tokyo 135-0064, Japan

Edited by Richard Henderson, Medical Research Council, Cambridge, United Kingdom, and approved May 7, 2008 (received for review March 23, 2008) Prostaglandins (PG) are bioactive lipids produced from arachidonic protein expression in this context seems resistant to anti-TNF␣ acid via the action of cyclooxygenases and terminal PG synthases. treatment or oral administration of corticosteroids (12, 13). Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an Together, these findings suggest that MPGES1 is a potential inducible glutathione-dependent integral membrane protein that target for development of therapeutic agents for the treatment catalyzes the oxidoreduction of cyclooxygenase derived PGH2 into of several diseases (10). PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and Results pain, and is therefore regarded as a primary target for develop- Structural Determination. We have determined the structure of ment of novel antiinflammatory drugs. To provide a structural human MPGES1 in complex with the tripeptide ␥-L-glutamyl- basis for insight in the catalytic mechanism, we determined the L-cysteinyl-glycine, glutathione (GSH) at 3.5 Å in-plane resolu- structure of MPGES1 in complex with glutathione by electron tion using electron crystallography (Table S1). The protein, crystallography from 2D crystals induced in the presence of phos- fused to an N-terminal His6-tag, was overexpressed in Esche- pholipids. Together with results from site-directed mutagenesis richia coli and purified in a Triton X-100 solubilized form. 2D and activity measurements, we can thereby demonstrate the role crystals were grown by dialysis in the presence of added phos- of specific amino acid residues. Glutathione is found to bind in a pholipids at a low lipid to protein ratio. The crystals often U-shaped conformation at the interface between subunits in the appeared as rounded rectangular sheets extending several ␮min protein trimer. It is exposed to a site facing the lipid bilayer, which the long dimension and Ϸ1 ␮m across (Fig. S2A). In the forms the specific environment for the oxidoreduction of PGH2 to crystalline sheets, the protein molecules were tightly packed and PGE2 after displacement of the cytoplasmic half of the N-terminal tilted 20° relative to the normal of the layer (Fig. S3). This transmembrane helix. Hence, insight into the dynamic behavior of arrangement explained an earlier observation that no end on MPGES1 and homologous membrane proteins in inflammation and views revealing the positions of transmembrane helices were detoxification is provided. observed from 0° projection maps (Fig. S2A) as had been demonstrated earlier for the MAPEG members MGST1 (14, 15) electron crystallography ͉ inflammation ͉ MAPEG ͉ membrane protein and LTC4S (16). Polar interactions within one unit cell involving arginine residues with exposed side chains at either the lumenal icrosomal prostaglandin E synthase 1 (MPGES1) is the or the cytoplasmic face of the protein were found to play Mkey enzyme in pathology related production of PGE2 from pertinent roles for crystal contacts (Fig. S3). cyclooxygenase (Cox) derived PGH2 (1). The protein is a member of the MAPEG protein family, which includes 5-lipoxy- Overall Structure. The subunits of MPGES1 form a homotrimer genase activating protein (FLAP), leukotriene C4 synthase (Fig. 1) in a similar way as for the other structurally characterized (LTC4S), microsomal glutathione transferase (MGST)1, MAPEG members, MGST1 (17), FLAP (18), and LTC4S (19, MGST2, and MGST3 (2, 3). MPGES1 is the most efficient PGES 20) and in agreement with earlier low resolution and hydrody- known and catalyzes the oxidoreduction of prostaglandin endo- namic data on MPGES1 (5). Our result thus supports the Ϫ1 Ϫ1 peroxide H2 into PGE2 with an apparent kcat/Km of 310 mM s suggestion that a trimeric arrangement is common to all [supporting information (SI) Fig. S1]. The enzyme equally well MAPEG proteins (21). Because the 2D crystals of MPGES1 are catalyses the oxidoreduction of endocannabinoids into prosta- orthorhombic the symmetry component within the trimer is a glandin glycerol esters (4) and PGG2 into 15-hydroperoxy-PGE2 local pseudo threefold axis. The variations in the immediate (5). In addition, the enzyme confers low glutathione transferase surroundings of the subunits in the asymmetric units of the unit and glutathione-dependent peroxidase activities (5). The bio- logical significance of the latter activities remains unclear but is thought to reflect the close evolutionary distance to MGST1. Author contributions: C.J., P.-J.J., and H.H. designed research; C.J., S.-C.P., P.P., P.B., K.R.G., N.G., K.M., and H.H. performed research; C.J., S.-C.P., P.P., P.B., R.M., P.-J.J., and H.H. MPGES1 protein expression levels are in most cases low, and analyzed data; and C.J., R.M., P.-J.J., and H.H. wrote the paper. proinflammatory stimuli induce its cellular expression and ac- The authors declare no conflict of interest. tivity, which is prevented by corticosteroids (1, 6–8). The This article is a PNAS Direct Submission. predominant source of PGH2 seems derived from Cox-2, al- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, though Cox-1 may also contribute (9). Studies, mainly from www.pdb.org (PDB ID code 3DWW). disruption of the MPGES1 gene in mice, indicate key roles for †To whom correspondence may be addressed. E-mail: [email protected], caroline. MPGES1-generated PGE2 in pathological conditions such as [email protected], or [email protected]. chronic inflammation, pain, fever, anorexia, atherosclerosis, §Present address: Molecular and Health Technologies, Commonwealth Scientific and stroke and tumorigenesis (10). Recently, a role for MPGES1 in Industrial Research Organization, 343 Royal Parade, Parkville VIC 3052, Australia. regulating neonatal respiration was described in ref. 11. This article contains supporting information online at www.pnas.org/cgi/content/full/ MPGES1 has been shown to be overexpressed in rheumatic 0802894105/DCSupplemental. diseases such as rheumatoid arthritis and myositis and the © 2008 by The National Academy of Sciences of the USA

11110–11115 ͉ PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802894105 Downloaded by guest on September 27, 2021 Fig. 2. Intra- and intersubunit interactions in MPGES1. (A) Stabilization of the cytoplasmic face of transmembrane helices 2 and 3. (B) Interactions between the same helices close to the GSH binding site. (C) Stabilization of the trimer through the His-72–Glu-77 ion pair.

inner core structure, which narrows down into complete closure toward the lumenal side by aromatic side chains. The funnel shaped opening toward the opposite cytoplasmic side is partly covered by the three large connections between TM1s and TM2s. These domains protrude toward the loops between TM3 and TM4 of neighboring subunits.

Fig. 1. Overall structure of MPGES1. (A)2Fo Ϫ Fc map contoured at 1.2 ␴ as Essentially no charged side chains are found exposed toward seen from the side at the interface between two subunits. (B and C) The trimer the phospholipid bilayer for MPGES1 (Fig. S5). The side chain subunits have been colored in ribbon representation. (B) Top view from the of Lys-26 in the center of TM1 points toward the interior of the face corresponding to the cytoplasm when the protein is positioned in its structure and makes an ion pair with Asp-75 of TM2 in the same natural membrane environment. (C) Side view with the cytoplasmic side up. subunit. This interaction gives rise to a distortion and bending of The transmembrane helices of one subunit have been labeled at their C- TM1. Arg-110, located in the center of TM3, is not exposed to terminal ends. the lipid bilayer but rather involved in intermolecular contacts. Compared with MPGES1, the crystal structure of LTC4S has cell suggest local structural variations at least in the peripheral more exposed charged side chains (Fig. S5). parts of the trimers. Mutagenesis. Our present structure and effects of site-directed The His6-tagged N terminus located on the lumenal side of the membrane embedded protein is flexible and the determined mutagenesis on activity have identified several important intra- and intermolecular contacts (Fig. 2 and Table 1). The cytoplas- model starts at Pro-11. The model begins with a long transmem- BIOCHEMISTRY mic face of TM2 is crucial for GSH binding and is stabilized brane (TM) helix ending at Lys-41. It is followed by a relatively toward TM3 of the same subunit close to its loop connection to large cytoplasmic loop region. Sequence alignment of MAPEG TM4 (Fig. 2A). The polar side chain residues involved in this members (Fig. S4) shows that this domain is highly variable and interaction Glu-66, Arg-67, Arg-70, and Tyr-117 are highly it is thus unlikely to be crucial for the catalytic functions of the conserved in MAPEG (Fig. S4). Among the human members MAPEG enzymes. The second TM helix, similar in length as the MGST3 has substitutions to asparagine and cysteine at positions first one, spans residues 61–90. A proline residue at position 81 corresponding to 66 and 67, respectively, whereas the noncata- gives rise to a slight kink located in the lumenal half of this helix. lytic FLAP has a threonine instead of arginine at position 70. This proline is highly conserved throughout the MAPEG family Mutating Arg-67 to alanine in MPGES1 completely abolishes but a shift in position between MPGES1/MGST1 and LTC4S/ activity (Table 1). Further stabilization toward the center of the FLAP (Fig. S4) gives rise to differences in helical arrangements. structure is achieved through side chains interactions from A short loop on the lumenal side connects TM2 with TM3. Both Asn-74, Glu-77 and Thr-78 of TM2 to Arg-110 and His-113 of TM3 (Pro-96-Gly-119) and TM4 (Pro-124-His-151), connected TM3 (Fig. 2B). The arginine residue at position 110 is conserved by a four-residue loop, are less exposed on the cytoplasmic side among all MAPEG members. Both Glu-77 and Arg-110 are of the membrane than the N-terminal helices. In the trimer the essential for MPGES1 catalysis, whereas the His72Ala mutant N terminus of one subunit is in close proximity to the C terminus shows some activity (Table 1). Toward the lumenal side of the of one of its neighbors. The three TM2s of the trimer form an trimer phenylalanines, tyrosines, and methionines are in close

Jegerscho¨ld et al. PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11111 Downloaded by guest on September 27, 2021 Table 1. Site-directed mutagenesis for MPGES1 Fraction of wild-type Mutation activity,* %

E66A 53 R67A 0 R70A 58 R70S 68 R70S† Full activity H72A 32 E77A 0 R110A 0 R110S 0 R110S† Lost activity Y117A 1 Y117F Full activity R70A-Y117A 7 Y130I‡ 15

Shown are residues identified to be involved in stabilisation of the protein and in GSH binding. *Activities were measured from isolated E. coli membranes (n ϭ 2). Values represent the percentage of the mean value from wt MPGES1 in isolated E. coli membranes (n ϭ 6). †From ref. 7. ‡From ref. 35. Fig. 3. Binding of GSH. (A) Top view of the MPGES1 trimer from the cytoplasmic side with the omit map contoured at 2.5 ␴, showing the positions of GSH molecules. (B) The U-shaped GSH molecule in ball and stick represen- proximity contributing to aromatic side chain interactions within tation with the omit map density as seen from the side at the interface and between subunits. His-72 in one subunit forms a salt bridge between transmembrane helices 1 and 4 from two different subunits. (C) Side to Glu-77 in the neighboring subunit (Fig. 2C). This histidine chains involved in formation of the binding pocket for GSH. residue in MGST1/MPGES1 is substituted by a glutamine in LTC4S and MGST2, whereas position 77 is a glutamic or an aspartic acid in all MAPEGs. active site of MPGES1 located on the cytoplasmic side. In MPGES1, the corresponding helices 1 and 4 are closer to- Glutathione Binding. We have grown the crystals of MPGES1 in gether and do not allow access to GSH. Therefore, we the presence of GSH. An omit map calculated between the conclude that our MPGES1 structure represents a closed observed electron diffraction amplitudes and amplitudes cal- conformation of the enzyme. Because no alternate access path culated from the protein model showed three distinct densities to GSH is evident it follows that dynamic opening and closing in more or less identical positions relative to each protein of the active site involving helices 1 and 4 is required to monomer (Fig. 3A). These regions were U-shaped (Fig. 3B) accommodate PGH2 (Fig. 4). Hydrogen/deuterium exchange similar to what has been observed for GSH in LTC4S (19, 20) dynamics of the closely related MGST1 supports dynamic but located deeper and at a different angle. In the vicinity of flexibility in helix 1 (22) that is decreased when a hydrophobic the GSH binding pocket a number of conserved residues are ligand is present (23). Furthermore, in the structures of LTC4 found that are involved in GSH binding (Fig. 3C). One GSH synthase a detergent molecule occupies the V-shaped cleft molecule is fixed by interacting with TM1 and 2 from one between subunits, which was proposed to be the substrate subunit and with TM2, 3 and 4 from its neighbor. The two binding site (19, 20). To generate a model (24) of an open arginine/tyrosine pairs Arg-70/Tyr-117 and Arg-126/Tyr-130 structure for MPGES1, we used LTC4S as a template (Fig. 4 are in close contact to the glycine and cysteine moieties of A and B). It is apparent that helix 1 occludes the active site in GSH, respectively. Arg-70 and Arg-38 make salt bridges to the the closed conformation as it clashes with PGH2 modeled to carboxylates at either end of the bent GSH molecule. Site- contact GSH whereas the open conformation allows access directed mutagenesis showed that substitution of Arg-70 for an (Fig. 4A Inset). The salt bridge between Lys-26 and Asp-75 alanine or a serine preserves some enzymatic activity, whereas appears to act as a hinge for the displacement of the cyto- the Arg70Ala/Tyr117Ala double mutant lost almost all activity plasmic half of TM1, which in that part does not have any (Table 1). The thiol group of GSH is stabilized by Arg-126 (3.8 strong interactions with neighboring helices. The open form of Å) and directed toward the membrane between helices 1 and MPGES1, resembling LTC4S regarding substrate access, thus 4 of neighboring subunits. constitutes a model for the productive enzyme. We believe that the closed state is in rapid equilibrium with an open state that Discussion is catalytically competent and very efficiently pulls the equi- Open/Closed Conformation of MPGES1. In LTC4S an opening librium in favor of product formation. In the resting enzyme between helix 1 in one subunit and helix 4 in a neighboring the presumed GSH thiolate could in this way be protected from subunit forms a V-shaped cleft that allows access to GSH from oxidation or unwanted side reactions. (The enzyme does the interior of the membrane (19, 20). This represents a natural display low glutathione transferase and peroxidase activity.) entry point for labile hydrophobic substrates, such as PGH2 and LTA4 that, by way of membrane entry, are protected from Mechanism. Two chemical mechanisms have been suggested for hydrolytic degradation. In addition, PGH2 is produced by PGE2 synthesis (25) where one involves attack of a thiol on the cyclooxygenases located on the lumenal side of the endoplas- O9 of the endoperoxide bridge in a glutathione peroxidase like matic reticulum and diffuses through the membrane to the mechanism. Because MPGES1 also catalyses GSH-dependent

11112 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802894105 Jegerscho¨ldet al. Downloaded by guest on September 27, 2021 Fig. 4. Conformational change allowing PGH2 access and schematic chemical mechanism for MPGES1. (A Left) Surface representation of the MPGES1 trimer structure as determined from the 2D crystals, showing a closed conformation. The three subunits are green, red, and blue, respectively. (A Right) Open conformation based on homology modeling from the structure of LTC4S (PDB entry 2UUH) exposes the GSH molecules. (Inset) Enlargement of the cleft between subunits where PGH2 is expected to bind. GSH is shown in yellow and a manually fitted PGH2 molecule shown in cyan. The surface rendering shows electrostatic potential of the open homology model. Blue represents positively charged, red represents negatively charged, and white represents a nonpolar surface. (B)(Left) The largest difference between the closed and open conformation is a bending of the cytoplasmic half of TM1 about a hinge fixed by the Lys-26/Asp-75 salt bridge (arrow). (Center) The closed conformation with Ser-127, Tyr-130, Thr-131, and Gln-134 onTM4 pointing toward residues Ile-32, Gln-34, and Leu-39 in TM1 in the next subunit. Pointing inwards binding to the GSH in magenta are Arg-126 from TM4 and Tyr-28 from TM1. (Right) The open conformation model with the same residues shown and with GSH in yellow. (C) Suggested chemical mechanism for the MPGES1 catalyzed PGE2 synthesis involves an attack of the GSH thiolate on BIOCHEMISTRY the O9 of the endoperoxide bridge followed by proton donation to O11 via Arg-126. Arg-126 then abstracts a proton from C9 where a carbonyl forms as the oxygen sulfur bond is broken. The leaving GSH thiolate is stabilized by Arg-126.

peroxidase activity toward other (hydro)peroxides, we favor mechanism is consistent with in vitro mutagenesis data sup- this mechanism that requires attack of the GSH thiolate, porting the GSH binding site location in MPGES1 (Table 1). stabilized by Arg-126 on the O9 oxygen of PGH2 (Fig. 4C). Alternatively, tyrosine(s) alone or in combination with argi- Strict restrictions are put on binding of PGH2 so that the nine could catalyze the chemistry. correct oxygen is attacked. A proton donor and acceptor are The structure of MPGES1 is an attractive target for devel- then required to assist in the chemical conversion (Fig. 4C) and oping drugs that could stabilize the closed form and thereby in a radius of 8 Å around the GSH sulfur only Arg-126 and efficiently inhibit catalysis. In addition, LTC4S could also exist tyrosines 28 and 130 are reasonable candidates for protonation in a closed conformation that was not experimentally acces- of O11 and proton abstraction from C9. Arg-126 could stabilize sible as the detergent used in crystallization behaved as a the evolving oxyanion O11 and donate a proton as the endo- substrate analogue (19, 20). The position of the hydrophobic peroxide is opened. This would allow Arg-126 to abstract a substrate in LTC4S and the similar location of GSH in proton (26) from C9 forming the carbonyl as the oxygen sulfur MPGES1/LTC4S show that PGH2 binds to the cleft between bond is broken regenerating the GSH thiolate (Fig. 4C). This TM1 and TM4. We have modeled a tentative binding mode,

Jegerscho¨ld et al. PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11113 Downloaded by guest on September 27, 2021 using the molecular ruler concept that places the omega-end We used the trimeric model of microsomal glutathione transferase 1 of PGH2 toward the middle of the bilayer, lets the endoper- (MGST1) (17) for a molecular replacement search, because the sequence oxide reside in proximity to GSH, and allows the carboxylate identity is high (Ϸ40%), and our studies have shown that MPGES1 also to reside in the hydrophilic protein water interface (Fig. 4A forms a trimer in 2D crystals (5). One of the top score solutions from AMoRe Inset). This tentative model suggests reasonable hydrogen (29) was unique, because it did not present severe clashes between trimers bonding and polar interactions allowing the hydrocarbon part and it fitted a low resolution projection map. Model building in O (30) to be bound along the cleft without steric clashes, which should started from a polyalanine model of the protein at the determined position and orientation. Numerous rounds of rigid body and restrained refine- be confirmed by the crystal structure of MPGES1 with a ment, using tight geometry restraints and medium noncrystallographic ligand. symmetry in REFMAC5 in combination with geometry idealization and manual rebuilding in O, resulted in a final structure of the MPGES1 trimer Materials and Methods in which all residues except the His-tags and the first 10 N-terminal residues Human MPGES1 containing a His6-tag at the N terminus was overexpressed in were included. At the present resolution, the difference between using E. coli and purified as published in ref. 5 and finally stored at a concentration electron- or x-ray form factors are negligible; thus, x-ray values were used of 1 mg/ml in a buffer containing 50 mM NaPi (pH 7.5), 50 mM NaCl, 10% throughout the refinement. 2Fo Ϫ Fc and Fo Ϫ Fc maps were continuously glycerol, 1 mM GSH, 1% Triton X-100, and 0.1 mM EDTA. generated to evaluate the accuracy and quality of the refined model. For 2D crystallization, we mixed aliquots of typically 50–100 ␮lofHis6- Reliable Rfree values were obtained by using a final fraction of Ϸ5% of the MPGES1 with bovine liver lecithin in Triton X-100 at a molar lipid to protein observed structure factor amplitudes. PROCHECK (31) was used to examine ratio of 9. The mixture was subjected to dialysis against the same buffer as the the molecular geometry. For analysis and illustrations, we used PyMOL (32) one used for protein storage but with 20% glycerol and lacking the detergent. and CHIMERA (33). Dialysis, using membranes with a cut-off molecular mass of 12–14 kDa, was Sequence alignment was performed with ClustalW (34) followed by struc- performed for at least 1 week at room temperature. For electron microscopy, we prepared grids with the inverted technique tural based manual adjustments. MODELLER 8, Version 2 (24) was used to (27), using conventional 400 mesh grids, thin carbon layers, typically 7% calculate homology models. trehalose and 2 ␮l of the crystallization suspension. Grids were placed in liquid The plasmid pSP19T7LT containing wild-type MPGES1 with an N- nitrogen cooled Gatan 626 or 914 cryoholders before insertion into a JEOL terminal His6-tag (5) was subjected to site-directed mutagenesis using 2100F electron microscope equipped with a TemCam-F415 4k ϫ 4k CCD GeneTailor site-directed mutagenesis system (Invitrogen) and specific mu- camera (Tietz Video and Image Processing Systems). tation primers. Mutant protein was expressed in BL21 Star (DE) E. coli cells Electron diffraction patterns were recorded on the CCD with two times grown at 37°C in terrific broth. Subsequently, we isolated the membrane binning at a camera length of 200 cm (Fig. S2B). For tilted diffraction patterns, fractions by differential centrifugation and quantified the amount of pairwise acquisitions were always made in such a way that the first tilted mutant MPGES1 by comparing the signals with known amounts of purified recording was followed by a 0° pattern that was used entirely for classification MPGES1 in Western blot analysis. For activity measurements, we used and quality assessment. By applying this approach of nontilted diffraction accordingly 400 ng of mutant or wild-type MPGES1 in the assay described pattern based sorting, we solved the problem of substantial crystalline het- in ref. 6. erogeneity and acquired a final set of 100 diffraction patterns showing good merging statistics (Table S1). Background corrected amplitudes were ex- ACKNOWLEDGMENTS. This work was supported by the Swedish Research tracted using the MRC software (28). The diffraction dataset was subjected to Council, Vinnova (Swedish Governmental Agency for Innovation Systems), the merging using DIFFMERG, and, subsequently, LATLINE was used for lattice line Japan Science and Technology Agency, the Swedish Cancer Society, and funds fitting. from Karolinska Institutet.

1. Jakobsson P-J, Thoren S, Morgenstern R, Samuelsson B (1999) Identification of human 14. Hebert H, et al. (1997) The 3.0 Å projection structure of microsomal glutathione prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, transferase as determined by electron crystallography of P21212 two-dimensional constituting a potential novel drug target. Proc Natl Acad Sci USA 96:7220–7225. crystals. J Mol Biol 271:751–758. 2. Jakobsson P-J, Morgenstern R, Mancini J, Ford-Hutchinson A, Persson B (1999) Common 15. Schmidt-Krey I, et al. (1999) The projection structure of the membrane protein micro- structural features of MAPEG—a widespread superfamily of membrane associated somal glutathione transferase at 3 Å resolution as determined from two-dimensional proteins with highly divergent functions in eicosanoid and glutathione metabolism. hexagonal crystals. J Mol Biol 288:243–253. Protein Sci 8:689–692. 16. Schmidt-Krey I, et al. (2004) Human leukotriene C4 synthase at 4.5 Å resolution in 3. Bresell A, et al. (2005) Bioinformatic and enzymatic characterization of the MAPEG projection. Structure 12:2009–2014. superfamily. FEBS J 272:1688–1703. 17. Holm PJ, et al. (2006) Structural basis for detoxification and oxidative stress protection 4. Kozak KR, et al. (2002) Metabolism of the endocannabinoids, 2-arachidonylglycerol in membranes. J Mol Biol 360:934–945. and anandamide, into prostaglandin, thromboxane, and prostacyclin glycerol esters 18. Ferguson AD, et al. (2007) Crystal structure of inhibitor-bound human 5-lipoxygenase- and ethanolamides. J Biol Chem 277:44877–44885. activating protein. Science 317:510–512. 5. Thoren S, et al. (2003) Human microsomal prostaglandin E synthase-1: Purification, 19. Ago H, et al. (2007) Crystal structure of a human membrane protein involved in functional characterization, and projection structure determination. J Biol Chem cysteinyl leukotriene biosynthesis. Nature 448:609–612. 278:22199–22209. 20. Martinez-Molina D, et al. (2007) Structural basis for synthesis of inflammatory medi- 6. Thoren S, Jakobsson P-J (2000) Coordinate up- and down-regulation of glutathione- ators by human leukotriene C4 synthase. Nature 448:613–616. dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by 21. Hebert H, Jegerscho¨ld C (2007) The structure of membrane associated proteins in NS-398 and leukotriene C4. Eur J Biochem 267:6428–6434. eicosanoid and glutathione metabolism as determined by electron crystallography. 7. Murakami M, et al. (2000) Regulation of prostaglandin E2 biosynthesis by inducible Curr Opin Struct Biol 17:396–404. membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxy- 22. Busenlehner LS, et al. (2004) Stress sensor triggers conformational response of the genase-2. J Biol Chem 275:32783–32792. integral membrane protein microsomal glutathione transferase 1. Biochemistry 8. Stichtenoth DO, et al. (2001) Microsomal prostaglandin E synthase is regulated by 43:11145–11152. proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells. 23. Busenlehner LS, et al. (2007) Location of substrate binding sites within the integral J Immunol 167:469–474. membrane protein microsomal glutathione transferase-1. Biochemistry 46:2812– 9. Ueno N, Takegoshi Y, Kamei D, Kudo I, Murakami M (2005) Coupling between 2822. cyclooxygenases and terminal prostanoid synthases. Biochem Biophys Res Commun 24. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial 338:70–76. restraints. J Mol Biol 234:779–815. 10. Samuelsson B, Morgenstern R, Jakobsson P-J (2007) Membrane prostaglandin E syn- 25. Yamada T, Komoto J, Watanabe K, Ohmiya Y, Takusagawa F (2005) Crystal structure thase-1: A novel therapeutic target. Pharmacol Rev 59:207–224. and possible catalytic mechanism of microsomal prostaglandin E synthase type 2 11. Hofstetter AO, Saha S, Siljehav V, Jakobsson P-J, Herlenius E (2007) The induced (mPGES-2). J Mol Biol 348:1163–1176. prostaglandin E2 pathway is a key regulator of the respiratory response to infection 26. Guillen-Schlippe YV, Hedstrom L (2005) A twisted base? The role of arginine in and hypoxia in neonates. Proc Natl Acad Sci USA 104:9894–9899. enzyme-catalyzed proton abstractions. Arch Biochem Biophys 433:266–278. 12. Korotkova M, et al. (December 18, 2007) Effects of immunosuppressive treatment on 27. Hirai T, Murata K, Mitsuoka K, Kimura Y, Fujiyoshi Y (1999) Trehalose embedding microsomal PGE synthase 1 and cyclooxygenases expression in muscle tissue of patients technique for high-resolution electron crystallography: Application to structural study with polymyositis or dermatomyositis. Ann Rheum Dis, 10.1136/ard.2007.079525. on bacteriorhodopsin. J Electron Microsc 48:653–658. 13. Korotkova M, et al. (2005) Effects of antirheumatic treatments on the prostaglandin E2 28. Crowther RA, Henderson R, Smith JM (1996) MRC Image processing programs. J Struct biosynthetic pathway. Arthritis Rheum 52:3439–3447. Biol 116:9–16.

11114 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802894105 Jegerscho¨ldet al. Downloaded by guest on September 27, 2021 29. Navaza J (1994) AMoRe: An automated package for molecular replacement. Acta 33. Pettersen EF, et al. (2004) UCSF Chimera—A visualization system for exploratory Crystallogr A 50:157–163. research and analysis. J Comput Chem 25:1605–1612. 30. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building 34. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity protein models in electron density maps and the location of errors in these models. of progressive multiple sequence alignment through sequence weighting, posi- Acta Crystallogr A 47:110–119. tion-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673– 31. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: A program to 4680. check the stereochemical quality of protein structures. J Appl Cryst 26:283–291. 35. Huang X, et al. (2006) Structural and functional characterization of human microsomal 32. De Lano WL (2002) The PyMOL Molecular Graphics System. Available at www.py- prostaglandin E synthase-1 by computational modeling and site-directed mutagenesis. mol.org. Accessed September 1, 2007. Bioorg Med Chem 14:3553–3562. BIOCHEMISTRY

Jegerscho¨ld et al. PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11115 Downloaded by guest on September 27, 2021